Effect of Buyang Huanwu Tang on LPS-induced Macrophage Activation and Autophagy / 中国实验方剂学杂志

ABSTRACT

Objective:To investigate the effect of Buyang Huanwu Tang in resisting lipopolysaccharide (LPS)-induced macrophage activation and autophagy through phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/Akt/mTOR) signaling pathway. Method:The cell counting kit-8 (CCK-8) method was used to screen out the optimal LPS concentration for inducing the activity of RAW264.7 macrophages. RAW264.7 macrophages were treated separately with PI3K blocker 3-methyladenine(3-MA) (5 mmol·L-1), Akt blocker MK2206 (5 μmol·L-1), mTOR blocker Rapamycin (10 μmol·L-1), Beclin1 blocker Spautin-1 (5 μmol·L-1), different doses of Buyang Huanwu Tang serum (5%, 10%, 20%) and the optimum concentration of LPS for 24 h. The concentrations of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in RAW264.7 macrophages were detected by enzyme-lined immunosorbent assay(ELISA). Western blot was used to detect the expression levels of phosphorylated PI3K, phosphorylated Akt, phosphorylated mTOR protein, microtubule light chain protein 3 (LC3), ubiquitin-binding protein 1 (p62) and Beclin-1. The autophagy flow of RAW264.7 cells was detected by transfection with autophagy double-labeled adenovirus. Result:Results of CCK-8 showed the highest cell viability when 10 mg·L-1 LPS was applied. The concentrations of IL-1β, IL-6 and TNF-α in the model group were significantly higher than those in the blank group (PPβ, IL-6 and TNF-α (PPPPPPPPConclusion:Buyang Huanwu Tang can resist LPS-induced macrophages activation and autophagy, inhibit macrophage inflammatory response, regulate PI3K/Akt/mTOR signaling pathway, and inhibit the excessive occurrence of autophagy.